Project Summary/Abstract Craniofacial bone tissue engineering has been extensively accomplished using bone grafting procedures. Mesenchymal stem cells (MSCs) are promising alternative treatment modality for bone regeneration. MSCs derived from orofacial tissues (e.g. gingival mesenchymal stem cells (GMSCs)) are attractive postnatal stem cells with self-renewal and multilineage differentiation capacities and superior osteogenic properties compared to bone marrow mesenchymal stem cells (BMMSCs). It is well known that biomaterials can be used to direct the fate of stem cells. However, controlling the fate of the transplanted stem cells is still a major challenge. Understanding the factors influencing the fate of encapsulating MSCs is of major therapeutic interest. Physiomechanical properties such as elasticity of the hydrogel biomaterial have been shown to be a vital factor influencing MSC differentiation, but their role on the MSC-host immune system is fairly unknown. To develop effective MSC- based regenerative therapies it is crucial to have a clear understanding of how the encapsulating hydrogel biomaterial elasticity affect the MSCs-host immune system interplay and immunoregulatory properties of MSCs. Therefore, the main objectives of this proposal are to (1) to explore the role of the elasticity of alginate hydrogel, as an encapsulating scaffold, in MSCs-T cells interplay and the detailed mechanism underlying the MSC immunomodulation.; and (2) To Investigate the role of elasticity of the encapsulating biomaterial on MSC- T cell interplay in the MSC-mediated bone tissue engineering. The central hypothesis of this proposal is that the elasticity of alginate hydrogel regulates the MSCs-host immune cells (T-cells)/ cytokines interplay, therefore, direct the fate of the encapsulated MSCs. Also, it is hypothesized that hydrogel elasticity can control the immunoregulatory function of the encapsulated MSCs, therefore, further regulates the microenvironment. Upon successful completion of the Specific Aims, this project will improve our understanding of the critical role of the biomaterials physiomechanical properties on MSC survival and fate determination. Moreover, it significantly improves our knowledge on how the matrix elasticity modulates MSCs' immunomodulatory properties.